Adiponectin is a secretory protein that is specifically expressed in adipocytes, and was reported to exhibit an anti-arteriosclerotic effect and an insulin resistance-reducing effect. In recent years, adiponectin has been reported to be involved in cancer, inflammation, and metabolic syndrome, and is considered to function as a defensive factor in the living body. Human adiponectin consists of 244 amino acid residues, and is known to have a molecular weight of approximately 28 kDa as a monomer. It was confirmed that adiponectin is present in blood in forms of a trimer (LMW: Low-molecular weight) as the basic structure, a hexamer (MMW: Middle-molecular weight) composed of the trimer, and multimers (HMW: High-molecular weight) composed of one or more LMWs and/or MMWs.
These various forms of adiponectin exhibit different physiological effects. For example, it was reported that HMW and MMW activated NF-κB in a C2C12 cell line, but LMW did not. Further, it was reported that persons deficient in adiponectin secretion could not produce HMW adiponectin and suffered hypoadiponectinemia and diabetes, and that the content ratio of HMW adiponectin was decreased in patients with coronary artery disease in comparison with healthy persons. Furthermore, it was reported that adiponectin of which the blood level changed before and after a diet was HMW. Therefore, it is important to detect HMW adiponectin alone in studies of adiponectin and various diseases.
In general, a protein molecule can be detected using a monoclonal antibody that specifically recognizes the protein as an antigen. However, in the case that the antigen is a mixture of multimers in various forms composed of single monomers such as adiponectin, it is difficult to obtain an antibody specific to only a molecule having a specific size. Because an antibody usually recognizes a primary amino acid sequence of an antigen, when an antigen is multimers, the antibody generally recognizes a primary amino acid sequence of the monomer that forms the multimers. Because adiponectin is composed of single monomers, an antibody that recognizes the adiponectin monomer simultaneously recognizes adiponectin multimers in any form. Even if a monoclonal antibody recognizes a tertiary structure, the crossreactivity to other molecules cannot be avoided in a molecule such as HMW adiponectin in which many basic structures are polymerized.
Therefore, in a case of using a general antibody, it is difficult to detect an adiponectin molecule having a specific size alone.
To quantitatively determine adiponectin having a specific molecular size, as conventional methods, a method in which adiponectin is pretreated (for example, gel filtration) to fractionate it based on its molecular weight and each peak is detected, and a method in which a specific molecule(s) is digested and the remaining molecule is detected, are generally used. Further, an ELISA technique that does not need pretreatment, such as gel filtration or a protease treatment, was developed, but crossreactivity to MMW is observed, and the ELISA technique is not specific to HMW. Therefore, a technique capable of detecting adiponectin having a specific molecular size alone, by a simple pretreatment or without pretreatment, is desired.
As conventional methods for measuring adiponectin having a specific molecular size, in addition to the above-mentioned gel filtration method, the following two methods are known:
(1) a method in which a pretreatment with an enzyme that digests LMW and MMW is carried out, and the remaining adiponectin that is not digested by the enzyme is measured to detect HMW alone (Patent literature 1), and
(2) a method in which an antibody that does not react with the adiponectin monomer, but recognizes a trimer and/or its associating molecule, high-molecular weight adiponectin, is used to detect the high-molecular weight adiponectin antigen alone (Patent literature 2).
However, the pretreatment with the enzyme needs an enzyme treatment time of several tens of minutes. In addition, in the case of incubation for a long time that exceeds an optimum time, there is a possibility that the target molecule per se may be digested by a coexisting protease, and thus, it is necessary to strictly select the incubation time. Further, in the treatment with an enzyme, an optimum digestive enzyme should be selected in accordance with the type of a target antigen, and thus, it is not a versatile method.
In the case of using the conventional antibody that recognizes high-molecular weight adiponectin, not only high-molecular weight adiponectin (HMW) but also other multimers (LMW and MMW) are simultaneously recognized, and thus, it is not a method capable of specifically detecting HMW alone. An antibody that specifically recognizes HMW adiponectin alone is unknown.